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[Cloning, prokaryotic expression and functional analysis of pep gene from Bacillus thuringiensis lysogenic
Wei Liao1, Weichun Chen, Yanhua Jia
1State Key Laboratory of Biocontrol and Institute of Entomology, Sun Yat-sen University, Guangzhou 510275, China. lw@gxzjy.com
Objective:
Random outbreak of lysogenic bacteriophage from Bacillus thuringiensis was very harmful to the production of Bacillus thuringiensis insecticide. We clarified the background of the phage from Bacillus thuringiensis MZ1 at the molecular level to solve the problem of random outbreak.
Methods:
After the strain MZ1 from a company in Meixian County of Guangdong Province was induced, we obtained phage particles. Then phage DNA was extracted and pep gene was cloned, expressed and analyzed.
Results:
We obtained a lysogenic bacteriophage MZTP01with clear plaque and 1 mm diameter. Fragment D with 2362bp (Genebank No. AY639599) was obtained after the phage DNA hydrolyzed by HindIII/EcoR I. Among fragment D, the pep gene with molecular weight of 47 kDa and length of 1101bp was cloned and expressed. Recombinant M15 (pQE30pep) was built and overexpressed in Escherichia coli with a 47kDa clear band. At the same place a clear band was observed by Western blot. Judging from the time course expression, we could conclude that PEP protein produced at 1 hour after induction and then increased gradually. PEP protein was mainly in the form of inclusion body in the recombinant and slowed the growth speed of host. Homologous comparison of PEP protein from phage MZTP01 with other PEPs from BLAST were that phage MZTP01 PEP protein had 100% homologe with that of Escherichia coli K12, and most of others took the similarity in the range between 37%-84%.
Conclusion:
PEP protein had ability to hydrolyze casein with the enzyme activity of 0.3 mg/ml trypsin. PEP protein may be a kind of trypsin.
Insights
Lysogenic bacteriophage outbreaks harm Bacillus thuringiensis production. Researchers identified and characterized the PEP gene from phage MZTP01, revealing its potential as a trypsin-like enzyme to address production issues.
Area of Science:
- Molecular biology
- Bacteriophage research
- Enzymology
Background:
- Lysogenic bacteriophage outbreaks pose a significant threat to Bacillus thuringiensis insecticide production.
- Understanding the molecular basis of phage behavior is crucial for mitigating these harmful outbreaks.
Purpose of the Study:
- To elucidate the molecular background of a specific bacteriophage from Bacillus thuringiensis MZ1.
- To identify and characterize genes responsible for phage activity, aiming to prevent random outbreaks.
Main Methods:
- Induction of Bacillus thuringiensis strain MZ1 to obtain phage particles.
- Extraction of phage DNA, followed by cloning, expression, and analysis of the pep gene.
- Overexpression of the pep gene in Escherichia coli and Western blot analysis.
Main Results:
- Isolation of lysogenic bacteriophage MZTP01 with a clear plaque.
- Cloning and successful expression of the pep gene (1101bp, 47 kDa) from phage MZTP01.
- PEP protein showed 100% homology with Escherichia coli K12 PEP and exhibited casein-hydrolyzing activity, suggesting a trypsin-like function.
Conclusions:
- The identified PEP protein possesses casein-hydrolyzing activity, indicating it is a type of trypsin.
- Characterization of PEP protein provides insights into bacteriophage mechanisms and potential control strategies.
